This application is a competing renewal of an R01 grant subsequently modified to R37 status following a Merit Award from NIDA. The initial application proposed to correlate the molecular properties of cannabimimetic agents with their biochemical/pharmacological properties in order to identify the pharmacophoric profiles of key compound classes that directly modulate the functions of the CB1 and CB2 cannabinoid receptors (CBRs). This information was used to design, synthesize and develop later generation CBR (ant) agonists with distinct pharmacological profiles either as molecular probes or leads for medications development. All of the above goals were completed. During this project period, we were motivated to extend our goals by new findings on the effects of enzymatic endocannabinoid deactivation by specific esterases. We now propose to study the three key brain esterases involved in endocannabinoid deactivation, namely, monoacyl glycerol lipase (MGL), fatty acid amide hydrolase (FAAH) and /-hydrolase domain-containing 6 (ABHD6) for their structural and functional properties. Inhibition of these enzymes enhances endocannabinoid levels, indirectly activates cannabinoid receptors and modulates cannabinergic signaling. All three enzymes have been implicated in mechanisms associated with addiction and pain and are potentially useful targets for therapeutic intervention. The proposed work will be carried out using ligands that interact covalently or non-covalently with each of the enzymes and will include mass spectrometry and nuclear magnetic resonance experiments to obtain detailed structural and functional information for each of the enzymes. The results will be used to design and synthesize selective reversible and irreversible inhibitors with optimized pharmacological profiles for each of the enzymes. Additionally, we propose to develop selective ligands possessing dual action with equal ability to inactivate two enzymes. Successful inhibitors will serve as useful pharmacological probes to study the endocannabinoid system as well as leads for the development of medications for addiction and pain management.